OP0224 LACK OF FOLYLPOLYGLUTAMATE SYNTHETASE ACTIVITY AND METHOTREXATE POLYGLUTAMYLATION AS UNDERLYING MECHANISMS EXPLAINING WHY METHOTREXATE DOES NOT IMPAIR SPERM QUALITY
Authors: L. F. Perez-Garcia, M. Lin, E. Röder, L. J. Kranenburg - van Koppen, B. Krijthe, R. Van Adrichem, E. Zirkzee, M. Van Doorn, G. Dolhe, P. Griffioen, E. Struys, G. Jansen, R. De Jonge, R. Dolhain
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> We have previously showed that exposure to methotrexate (MTX) was not associated with sperm quality abnormalities or with sperm DNA damage [1]. Nonetheless, a question that remains unanswered is whether MTX and its bioactive forms, the MTX-polyglutamates (MTX-PGs) can be found in spermatozoa. The intracellular conversion of MTX to MTX-PGs is dependent on a process that is catalyzed by the enzyme folylpolyglutamate synthetase (FPGS). Lower FPGS activity and subsequent inefficient polyglutamylation is well-known phenomenon associated with a rapid efflux of MTX from cells [2]. In this regard, recent studies have showed that a significant reduction of FPGS activity is associated with aberrant pre-mRNA splicing of this enzyme, including partial retention of intron 8 (8PR) and a higher ratio of FPGS 8PR over FPGS wild type (WT) [3]. Whether intracellular MTX and the bioactive MTX-PGs can be detected in the spermatozoa and whether spermatozoa have the enzymatic capabilities (FGPS-activity) for the formation of the MTX-PGs has never been studied before. <h3>Objectives</h3> To evaluate if MTX-PGs can be detected in spermatozoa of men exposed to MTX and to evaluate the FPGS enzymatic activity in spermatozoa. Furthermore, MTX-PGs concentration and FPGS activity in spermatozoa will be compared with those present in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs). <h3>Methods</h3> This proof of concept translational study was part of the iFAME-MTX study [1]. Blood and semen samples were obtained from 10 adult men diagnosed with an immune-mediated disease (RA, SpA, psoriasis) after 13 weeks of exposure to MTX (cases) and from 4 adult healthy men (controls). Measurement of MTX-PGs in spermatozoa and RBCs was determined by LC-MS/MS methods [4] using stable isotope labelled MTX-PGs<sub>1-6</sub> as internal standards. FPGS catalytic activity in spermatozoa, RBCs and PBMCs was analyzed in 10 μg protein extracts [3]. Lastly, RNA was isolated from spermatozoa and PBMCs quantitative real-time PCR were performed to quantify the mRNA expression profiles of folate genes in spermatozoa, in particular FPGS 8PR/WT [3]. Continuous variables were compared using a Mann-Whitney test. <h3>Results</h3> MTX-PG<sub>1</sub> was the predominant form of MTX-PGs detected in spermatozoa. Other MTX-PGs forms (MTX-PG<sub>2-5</sub>) were not or hardly detectable in spermatozoa after MTX exposure (Figure 1A). Furthermore, comparisons with other cell types revealed that MTX-PGs accumulation in spermatozoa is markedly lower than in RBCs and PBMCs (Figure 1B). Consistent with the lack of MTX-PGs formation, FPGS catalytic activity (reported as pmol 15N-MTX-PG2 formed/h/mg protein) in spermatozoa was statistically significant lower (5.1) compared to PBMCs (183, p=&lt;0.005)). Regarding, mRNA expression profiles of folate genes in spermatozoa, a high FPGS 8PR/WT ratio was observed compared to PBMCs. <h3>Conclusion</h3> This is the first study ever conducted to measure the concentration of MTX-PGs and concomitant FPGS activity in spermatozoa. Hardly any bioactive forms of MTX (MTX-PG) could be detected in spermatozoa. This is related to the fact that spermatozoa have very low FPGS activity and hence lack the capacity to form MTX-PGs. Mechanistically, the low FPGS activity in spermatozoa is likely associated with a higher ratio of mRNA expression of an alternatively spliced form of the FPGS gene (8PR) over the WT transcript. These results support and clarify our previous findings that treatment with MTX does not affect sperm quality parameters and provides further evidence that MTX can be safely used in men with a wish to become a father. <h3>References</h3> [1]Perez-Garcia LF, et al. Annals of the Rheumatic Diseases. 2022;81(Suppl 1):84-. [2]Rots MG, et al. Blood. 1999;93(5):1677-83. [3]Muller IB, et al. Rheumatology (Oxford). 2021;60(3):1273-81. [4]Hebing RC, et al. Ann Rheum Dis. 2022. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.